Appropriate enriched Vinasse culture medium. In this section, the behavior of S. pasteurii bacterium incubated within the considered culture media is assessed based on their growth curves. Fig 4 presents growth curves of S. pasteurii for different culture media. The experiments were conducted with three replications, and the growth curves were plotted based on the mean values of the data. The growth curves were drawn until the bacteria growth process entered the death phase in each culture medium. In fact, the growth curves exhibit all four possible phases, including the lag (delay), log (logarithmic), stationary, and death phase.
As seen in Fig 4, for the conventional culture medium (NB+U), the S. pasteurii bacterium enters the stationary phase approximately 10 hours after the beginning of growth, with a maximum number of grown bacteria of 109.3 per milliliter. In contrast, when using the proposed culture media, the bacterium achieved higher maximum reproductive rates, but entered the stationary phase over a longer period (around 14 hours). On the other hand, the maximum number of bacteria significantly increased to 109.7-1011.4.
The enriched vinasse culture media have been demonstrated to increase the maximum number of multiplied bacteria , i.e., more than 9.5 logarithmic units, to a greater extent than other waste materials such as corn steep liquor (less than 9 logarithmic units), lactose mother liquor (less than 8 logarithmic units), yeast extract (less than 8 logarithmic units) which were previously introduced as waste culture media for the S.pasteurii bacterium [32, 35, 37, 38, 67]. Additionally, the stationary phase in the proposed culture media lasts over 20 hours, which is significantly longer than the conventional culture medium's stationary phase of 10 hours. The longer stationary phase is particularly important for practical applications of the MICP method since it allows more time to introduce bacteria into the soil.
The higher number of bacteria in the culture medium can lead to more effective soil treatment due to the increased production of urease enzyme within the soil. Based on the growth curves, it appears that the V+U culture medium is the most effective one for growing S. pasteurii bacterium among other considered culture media. Nevertheless, it is worth noting that the differences in the growth curves between the various media are relatively small and the urease activity of the bacteria may play a crucial role to adopt the most appropriate culture medium for the MICP procedure. Therefore, additional tests were conducted to examine the urease activity of S. pasteurii bacterium grown within the different culture media.
As discussed previously, Urease activity was determined for S. pasteurii bacterium in the different culture media including N+U, V+NaCAS, V+WPC, and V+U, by measuring the amount of ammonia released from hydrolyzed urea using the Nessler method (at 425 nm, 25°C, pH 7). To identify the most appropriate culture medium, the urease activity of S. pasteurii bacterium was measured in different media in the middle of their stationary phase, i.e., 14 hours for conventional culture medium and 24 hours for the proposed culture media (Table 1).
Table 1. The result of urease activity tests for S. pasteurii bacterium in various culture media.
Culture media
|
NB+ U
|
V+ U
|
V+ NaCAS
|
V+ WPC
|
Urease activity (U/min)
|
216
|
447*
|
386
|
312
|
*Highest urease activity
|
|
|
|
|
The results of the urease activity tests show that the urease activity of S. pasteurii bacterium in the new culture media is significantly greater than NB+U medium while the highest value devotes to the V+U culture medium. Additionally, the use of enriched vinasse culture media has been shown to enhance the urease activity of S. pasteurii bacterium, i.e., 447 U/min, to a greater extent than waste materials such as corn steep liquor (less than 300 U/min), lactose mother liquor (less than 250 U/min), yeast extract (less than 270 U/min) which were previously introduced as culture media for the S.pasteurii bacterium [32, 35, 67].
For economic assessment purpose, the costs of the considered culture media were estimated by comparing the prices of all elements used in the proposed media in terms of the average industrial prices in Iran. The estimated costs are presented in Table 2.
Table 2. Estimated cost of different culture media considered for MICP treatment
Culture media
|
NB+U
|
V+U
|
V+NaCAS
|
V+WPC
|
Cost*/l (US$)
|
0.455
|
0.005
|
0.315
|
0.551
|
Relative cost in comparison to NB+U
|
1
|
0.011
|
0.692
|
1.210
|
*Approximate industrial grade price (current market)
|
Table 2 shows that the V+U culture medium has the lowest cost compared to the other alternatives, with costs up to 91 times lower than that of the conventional culture medium. Therefore, using the V+U culture medium can substantially reduce the initial costs of the MICP technique and increase its applicability.
This study found that the combination of vinasse and urea provides suitable conditions for the growth and production of the urease enzyme. The results in fact showed that urea was the most effective supplement for the growth of S. pasteurii bacterium in the culture medium containing vinasse. Previous research has also demonstrated that urea is the suitable supplement for the growth of the bacterium within other culture media [68, 69].
In summary, the use of urea-enriched vinasse (V+U) culture medium can significantly improve the efficiency of the MICP method by increasing both the urease activity and number of bacteria. Moreover, it is the most cost-effective culture medium among the considered alternatives. Hence, V+U culture medium is suggested as the best culture medium alternative and further tests are conducted to optimize this medium for soil improvement purposes.
Optimum culture medium. As highlighted in the preceding section, the utilization of vinasse as a fundamental constituent of bacterial culture medium in the MICP brings about an upsurge in the number of bacteria and urease activity, while simultaneously rendering the MICP process cost-effective. Optimizing the proportion of vinasse, which contains moderate levels of nitrogen, as well as other organic and inorganic elements, within the proposed culture medium is a crucial aspect of this study. To ascertain the ideal proportion of vinasse and urea in the culture medium, the study utilized TDOE, ANOVA, and ANOM to evaluate the bacterial count (i.e., log (cfu/ml)) and the urease activity of S. pasteurii (i.e., UA (U/min)). The study employed the L9 array to design the experiments, and based on the initial trials, it was found that the most suitable values for Vinasse Concentration, Incubation Time, and Urea Concentration lie between 4-10% l/l, 10-30 h, and 1.5-2.5% g/l, respectively. These values were determined to yield an optimal number of bacteria and urease activity.
Table 3 displays the logarithmic values of bacterial number and urease activity for the T1 to T9 experiments. Previous research has demonstrated that the TDOE method can predict outcomes with a satisfactory level of accuracy [13, 66, 70, 71]. Table 3 also includes the predictions of the TDOE method, which were calculated using the Minitab software package [72] for full factorial test conditions, as well as the S/N ratios obtained using Eq. (1).
Table 3. Comparison between the experimental results, confirmation tests and TDOE predictions
|
Factor level
|
|
log (cfu/ml)
|
|
UA (U/min)
|
|
V (%)
|
Time (h)
|
U (%)
|
|
Test
|
TDOE
|
S/N
|
|
Test
|
TDOE
|
S/N
|
T1
|
4
|
10
|
1.5
|
|
6.81
|
6.63
|
16.10
|
|
327.86
|
325.54
|
50.73
|
|
6.03
|
|
362.42
|
T2
|
4
|
20
|
2
|
|
9.29
|
8.57
|
19.89
|
|
323.59
|
308.96
|
50.47
|
|
10.59
|
|
344.71
|
T3
|
4
|
30
|
2.5
|
|
5.81
|
7.10
|
15.47
|
|
218.98
|
260.66
|
46.68
|
|
6.07
|
|
212.76
|
T4
|
7
|
10
|
2
|
|
6.24
|
7.74
|
16.33
|
|
247.98
|
299.97
|
48.13
|
|
6.92
|
|
262.38
|
T5
|
7
|
20
|
2.5
|
|
9.94
|
10.29
|
20.07
|
|
384.57
|
359.75
|
51.58
|
|
10.22
|
|
374.13
|
T6
|
7
|
30
|
1.5
|
|
11.56
|
10.35
|
21.38
|
|
431.65
|
401.61
|
52.60
|
|
11.88
|
|
421.95
|
T7
|
10
|
10
|
2.5
|
|
9.46
|
8.31
|
19.71
|
|
421.57
|
393.11
|
52.43
|
|
9.90
|
|
415.03
|
T8
|
10
|
20
|
1.5
|
|
11.10
|
12.41
|
21.02
|
|
513.45
|
543.03
|
53.94
|
|
11.40
|
|
483.03
|
T9
|
10
|
30
|
2
|
|
9.97
|
10.33
|
20.10
|
|
441.08
|
418.38
|
52.83
|
|
10.27
|
|
434.88
|
T10
|
4
|
10
|
2
|
|
-
|
5.71
|
-
|
|
-
|
224.58
|
-
|
T11*
|
4
|
10
|
2.5
|
|
-
|
5.40
|
-
|
|
-
|
239.99
|
-
|
T12
|
4
|
20
|
1.5
|
|
-
|
9.49
|
-
|
|
-
|
389.91
|
-
|
T13
|
4
|
20
|
2.5
|
|
-
|
8.26
|
-
|
|
-
|
304.36
|
-
|
T14*
|
4
|
30
|
1.5
|
|
-
|
8.33
|
-
|
|
-
|
346.22
|
-
|
T15
|
4
|
30
|
2
|
|
-
|
7.41
|
-
|
|
-
|
265.26
|
-
|
T16*
|
7
|
10
|
1.5
|
|
-
|
8.65
|
-
|
|
-
|
380.93
|
-
|
T17*
|
7
|
10
|
2.5
|
|
-
|
7.42
|
-
|
|
-
|
295.38
|
-
|
T18
|
7
|
20
|
1.5
|
|
-
|
11.52
|
-
|
|
-
|
445.30
|
-
|
T19
|
7
|
20
|
2
|
|
-
|
10.60
|
-
|
|
-
|
364.35
|
-
|
T20*
|
7
|
30
|
2
|
|
-
|
9.44
|
-
|
|
-
|
320.65
|
-
|
T21
|
7
|
30
|
2.5
|
|
-
|
9.12
|
-
|
|
-
|
316.05
|
-
|
T22
|
10
|
10
|
1.5
|
|
-
|
9.54
|
-
|
|
-
|
478.66
|
-
|
T23*
|
10
|
10
|
2
|
|
-
|
8.63
|
-
|
|
-
|
397.70
|
-
|
T24
|
10
|
20
|
2
|
|
-
|
11.49
|
-
|
|
-
|
462.08
|
-
|
T25
|
10
|
20
|
2.5
|
|
-
|
11.18
|
-
|
|
-
|
457.48
|
-
|
T26
|
10
|
30
|
1.5
|
|
-
|
11.24
|
-
|
|
-
|
499.34
|
-
|
T27*
|
10
|
30
|
2.5
|
|
-
|
10.04
|
-
|
|
-
|
413.78
|
-
|
Table 4 summarizes the results of ANOM as per Table 3 test results. According to Table 4 and considering the fact that larger values of number of bacteria and urease activity can cause better outcomes for microbial soil treatment purposes, the optimal levels for Vinasse concentration (V), incubation Time, and Urea concentration (U) are the third, second, and first levels, respectively. The ANOM analysis indicates that higher Vinasse concentration leads to increased levels of urease activity and number of bacteria, with 1.5 g/lit of urea being the most effective concentration for optimal culture medium. The optimal percentage of the urea is lower in the proposed culture medium, i.e., 1.5% compared to 2% considered in previous studies for S. pasteurii bacterium (Whiffin 2004, Babakhani et al. 2021, Achal et al. 2010, Achal et al. 2009, Stocks-Fischer et al. 1999). This variation can be attributed to the nitrogen content present in the Vinasse substance. The cost of waste culture media is contingent on the costs of supplements needed for bacterial growth. The proposed culture medium utilized in this study necessitates smaller quantities of urea as a supplement, rendering it a more cost-effective option. The findings indicate that the Vinasse concentration is the most influential parameter. Based on the ANOM, the optimal culture medium has a Vinasse concentration (V) of 10% and Urea concentration (U) of 1.5%, with an incubation time of 20 hours.
Table 4. Summary of ANOM results
|
Mean S/N Ratio
|
Significance of Parameter
|
|
log (cfu/ml)
|
|
Effecting Parameters
|
Level 1
|
Level 2
|
Level 3
|
Max-Min
|
V (%)
|
17.16
|
19.26
|
20.28*
|
3.12*
|
Time (h)
|
17.38
|
20.33*
|
18.98
|
1.60
|
U (%)
|
19.50*
|
18.77
|
18.42
|
1.08
|
|
UA (U/min)
|
|
V (%)
|
49.29
|
50.77
|
53.07*
|
3.77*
|
Time (h)
|
50.43
|
51.99*
|
50.70
|
1.57
|
U (%)
|
52.42*
|
50.47
|
50.23
|
1.95
|
* The most affecting parameter
|
Effects on soil microbial stabilization. To investigate the impact of using the optimal culture medium on bio-treated soil, a series of direct shear tests were conducted on loose and bio-treated sand specimens. The tests were performed using both the fresh optimal culture medium (V(100 ml/l)+U(15g/ml)) and the conventional culture medium (NB(3 g/l)+U (20 g/l)). The data obtained from the direct shear tests (Table 5) indicate a significant increase in the shear strength parameters of the treated soil when using the proposed culture medium. Compared to the untreated soil, the sample treated with the optimum culture medium exhibited an increase of 7.6° in the friction angle and 219.5 kPa in cohesion. On the other hand, the conventional culture medium resulted in an increase of 5.1° in the friction angle and 198.8 kPa in cohesion. This increase in shear strength parameters can be attributed to the higher number of bacteria and increased urease activity of the optimal culture medium. Additionally, vinasse, which is a calcium-containing biopolymer, also has a positive impact on soil stabilization as a soil stabilizer. To sum up, the use of the newly introduced culture medium for the MICP method will result in a noteworthy enhancement in the shear strength of soil samples. This improvement can be mainly attributed to the augmented cohesion between soil particles, as previously demonstrated in studies [9, 73, 74].
Table 5. Strength parameters of soil and MICP-treated soil using the proposed culture media and control one
Culture media
|
None(untreated)
|
NB (3 g/l)+U (20 g/l)
|
V (100 ml/l)+U (15 g/ml)
|
Friction angle ( )
|
31.0
|
36.1
|
38.6
|
Cohesion (kPa)
|
0
|
198.8
|
219.5
|
SEM images of soil samples were obtained to assess the quality of microbial calcite precipitation within soil pores and on the surface of soil particles following the bio-treatment. The SEM images of both untreated and treated soil samples are presented in Fig 5.
Upon examination of Fig. 5, it is evident that the use of new culture medium results in the thorough coating of sand particles, and the formation of calcite bridges between these particles is also noticeable. These bridges enhance the cohesion of the bio-treated soil medium.
Additionally, different types of calcium carbonate ions (e.g., calcite, aragonite, and vaterite) can be produced in an MICP process, but amongst these carbonates, calcite is the most stable one, hence is preferred when an MICP treatment is adopted [17, 40, 75] . To identify the type of calcium carbonates created in the MICP-treated soil samples of this study, the XRD technique was adopted. Fig 6 presents the XRD analysis of loose soil, and bio-treated samples by conventional and the proposed culture media. The results show that the calcium carbonate crystals in the sample by the conventional culture medium were mainly of calcite type, while these crystals were magnesium calcite crystals (i.e., Mg0.1Ca0.9CO3) in using the optimum V+U culture medium. The reason for the presence of magnesium in the calcium carbonate crystals is the result of using the new proposed culture medium and the present of magnesium elements in the vinasse substance. The maximum intensity of the crystals in both MICP treated samples were at the 2θ between 29.6° and 29.8° which is in agreement with several previous studies in this regard [17, 52, 76, 77]. Figure 6 shows that the characteristic diffraction peak of quarts was also detected, as expected considering that the soil used in this study was a quartz sand.
The results of direct shear tests, SEM imaging and XRD analysis suggest that S. pasteurii performs well in the new culture medium containing vinasse and urea for the MICP method. In other words, the new culture medium (V(100 ml/l)+U (15 g/l)) is a viable alternative to the conventional NB (3 g/l)+U (20g/l) culture medium for soil bio-treatment projects utilizing the MICP method.